This invention relates to a DNA molecule encoding a variant human xcex12B-adrenoceptor, said variant xcex12B-adrenoceptor protein and a method to assess the risk of individuals to suffer from vascular contraction of coronary arteries in mammals as well as a method for the treatment of vascular contraction of coronary arteries. This invention also relates to transgenic animals comprising a human DNA molecule encoding human xcex12B-adrenoceptor or said variant xcex12B-adrenoceptor.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The xcex12-adrenoceptors xcex12-ARs) mediate many of the physiological effects of the catecholamines norepinephrine and epinephrine. Three genetic subtypes of xcex12-adrenoceptors are known in humans and other mammals, denoted as xcex12A-, xcex12B-, and xcex12C-adrenoceptors. The human genes encoding the receptors are located on chromosomes 10, 2 and 4, respectively. No splice variants are known to exist of these receptors, as the genes are intronless. The tissue distributions and physiological and pharmacological functions of the receptor subtypes have been reviewed e.g. by MacDonald et al. (1997) and Docherty (1998). Based on recent studies with gene-targeted and transgenic mice, xcex12A-adrenoceptors mediate most of the pharmacological actions ascribed to currently available xcex12-adrenoceptor agonists, including inhibition of neurotransmitter release, central hypotensive and bradycardic effects, sedation and anesthesia, and analgesia. The same studies indicate that xcex12B-adrenoceptors mediate peripheral vasoconstriction in response to agonist activation (Link et al. 1996, Macmillan et al. 1996). Other physiological or pharmacological effects have not been associated with certainty with this receptor subtype. The xcex12C-adrenoceptor subtype appears to be involved in regulation of complex behaviors. It is not known that this subtype would have important functions in peripheral tissues outside the central nervous system or in cardiovascular regulation.
Coronary heart disease (CHD), like many other common disorders, arises from complex interactions between genetic and environmental factors. It is reasonable to assume that functionally important genetic variation in mechanisms important for the regulation of vascular functions, including the coronary vasculature, will be found to be associated with the pathogenesis and therapy of CHD. A variant form of the human xcex12B-AR gene was recently identified (Heinonen et al., 1999). The variant allele encodes a receptor protein with a deletion of three glutamate residues in an acidic stretch of 18 amino acids (of which 15 are glutamates) located in the third intracellular loop of the receptor polypeptide. This acidic stretch is a unique feature in the primary structure of xcex12B-AR in comparison to xcex12A-AR and xcex12C-AR, suggesting that the motif has a distinct role in the function of xcex12B-AR. Amino acid sequence alignment of xcex12B-AR polypeptides of different mammals reveals that the acidic stretch is highly conserved among the xcex12B-ARs of mammals and that the acidic stretch is long in humans in comparison to other species. This suggests that the motif is important for the functionality of the receptor, and that the short form (D for xe2x80x9cdeletionxe2x80x9d) probably represents the ancestral form and the long form (I for xe2x80x9cinsertionxe2x80x9d) could well represent a more recent allelic variant in humans. Jewell-Motz and Liggett (1995) studied the in vitro functions of this stretch using site-directed mutagenesis to delete as well as to substitute 16 amino acids of the stretch. Their results suggest that this acidic motif is necessary for full agonist-promoted receptor phosphorylation and desensitization.
Based on the vasoconstrictive property of xcex12B-AR in mice and the involvement of this acidic region in the desensitization mechanism of the receptor, we hypothesized that the deletion variant confers reduced receptor desensitization and therefore augmented vasoconstriction that could be associated with cardiovascular pathologies. To test this hypothesis, we carried out a 4-year prospective study in 912 middle-aged Finnish men.
One object of this invention is to provide a DNA sequence of a variant human xcex12B-adrenoceptor gene and the corresponding variant xcex12B-adrenoceptor protein.
Another object of the invention is to provide a method for screening a subject to assess if an individual is at risk to suffer from vascular contraction of coronary arteries.
A third object of the invention is to provide a method for the treatment of vascular contraction of coronary arteries of mammals.
A fourth object of the invention is to provide a transgenic animal with a gene encoding a human xcex12B-adrenoceptor or said variant thereof.
Thus, according to one aspect the invention concerns a DNA sequence comprising a nucleotide sequence encoding a variant xcex12B-adrenoceptor protein with a deletion of at least 1 glutamate from a glutamic acid repeat element of 12 glutamates, amino acids 298-309, in an acidic stretch of 18 amino acids 294-311, located in the 3rd intracellular loop of the receptor polypeptide.
The invention further concerns a variant xcex12B-adrenoceptor protein with a deletion of at least 1 glutamate from a glutamic acid repeat element of 12 glutamates, amino acids 298-309, in an acidic stretch of 18 amino acids 294-311, located in the 3rd intracellular loop of the receptor polypeptide.
According to another aspect the invention concerns a method for screening a subject to determine if said subject is a carrier of a said variant gene with both alleles encoding a said variant xcex12B-adrenoceptor, i.e. to determine if said subject""s genotype of the human xcex12B-adrenoceptor is of the deletion/deletion (D/D) type, comprising the steps of
a) providing a biological sample of the subject to be screened,
b) providing an assay for detecting in the biological sample the presence of
i) the insertion/insertion (I/I) or deletion/insertion (D/I) genotypes of the human xcex12B-adrenoceptor, or
ii) the D/D genotype of the human xcex12B-adrenoceptor, and
c) assessing at least one of the two following
i) an individual""s risk to develop a disease involving vascular contraction of coronary arteries, or
ii) an individual""s need for xcex12B-selective or xcex12B-nonselective xcex12-adrenoceptor antagonist therapy,
based on whether said subject is of said D/D genotype or not.
According to a third aspect the present invention concerns a method for treating a mammal suffering from vascular contraction of coronary arteries, said method comprising the step of administering a selective xcex12B-adrenoceptor antagonist to said mammal.
According to a fourth aspect the present invention concerns a transgenic animal which carries a human DNA sequence comprising a nucleotide sequence encoding a human xcex12B-adrenoceptor protein or a variant thereof.